Blocks in the pseudouridimycin pathway unlock hidden metabolites in the Streptomyces producer strain

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Metabolomic analysis of pum mutants

The functions of seven genes in the PUM gene cluster was previously assigned through bioinformatic analysis and the intermediates detected in knockout mutants [17], as summarized in Table 1. Briefly, the pseudouridine synthase PumJ catalyzes N– to C-nucleoside isomerization to yield pseudouridine or a derivative thereof, which is then converted into 5′-amino-5′-deoxy pseudourydine (APU) by the action of the oxidoreductase PumI and the aminotransferase PumG. In a converging step, guanidinoacetate (GAA) is produced by the amidino transferase PumN. Next, the amide ligases PumK and PumM add sequentially glutamine and GAA to APU, while PumE catalyzes N-hydroxylation (Table 1).

Table 1 pum mutants and accumulated intermediates. Concentrations (µM) of all intermediates except GAA were measured against a PUM standard. GAA concentrations expressed as ratios to the accumulation in the WT strain. The highest production of each metabolite is in bold type. See text for abbreviations. ND not detected.

In order to analyze globally the metabolome of pum knockout mutants, we cultivated the wild type producer strain, Streptomyces sp. ID38640 (WT) along with the seven previously reported knockout mutant strains (ΔpumE, ΔpumG, ΔpumI, ΔpumJ, ΔpumK, ΔpumM, and ΔpumN) and three newly constructed ones (ΔpumF, ΔpumH and ΔpumL; see below), all unable to produce PUM but accumulating different intermediates (Table 1). Each strain was cultivated in two media and we analyzed the metabolite distribution by LC–MS at 24-h intervals over four days. Samples were analyzed after solvent extraction of the whole culture (FE samples; usually hydrophobic metabolites) and also by direct analysis of the cleared broth (SN samples; both hydrophobic and hydrophilic metabolites). The LC–MS/MS data from 44 samples, derived from 11 strains cultivated in two different media with two samples per culture, were subjected to the Global Natural Products Social (GNPS) molecular networking analysis11 and visualized using Cytoscape18. This analysis clusters spectra having identical MS2 patterns, forming nodes (rectangles in Fig. 1) and connects nodes having fragmentation patterns sharing at least 4 fragments (lines in Fig. 1). This results in the formation of networks, representing families of potentially related metabolites. Analysis with Cytoscape provides an intuitive visualization of metabolite distribution according to strain, medium or sample.

Figure 1
figure1

Molecular network of samples from Streptomyces sp. ID38640 and ten knockout pum mutants. The analysis includes two cultures from each strain and two samples per culture. Node colors give the contributing medium: orange for M8, green for PumP1, light blue for nodes observed in both media. Black circles indicate PUM-related nodes, red circles indicate nodes corresponding to known compounds, green circles show unknown metabolites (NK). The associated BGCs, as listed in Table 2, are shown next to each metabolite.

The molecular network of Fig. 1, which was derived from 72-h samples, consists of 475 features, including media components, 369 of which are assigned to 36 molecular families, and 106 of which are singletons. Similar results were observed when analyzing samples at different time points, but the 72-h samples the highest metabolite richness. The two tested media afforded equivalent numbers of signals, each showing about 20% unique signals. Each pum knockout mutant strain contributed with 200–300 signals each, with ΔpumK and ΔpumH being at the upper and the lower end, respectively. Similarly, ~ 50% of signals were shared between the FE and SN samples, with ~ 25% unique signals contributed by each sample.

Correlating metabolites with biosynthetic gene clusters

From a draft genome of Streptomyces sp. ID38640, the genome mining tool antiSMASH 5.03 revealed 27 distinct regions harboring BGCs and a predicted metabolic diversity including five non-ribosomal peptides, five polyketides, seven ribosomally synthesized post translationally modified peptides, four terpenes, as well as at least nine regions classified as “other” (Table 2). Overall, 11 BGCs find a match with related BGCs in the MIBiG database.

Table 2 BGCs identified in ID38640 and presence of these BGCs in the four PUM producers.

Consistent with our previous work17, we detected molecular families corresponding to the lydicamycins and desferroxiamines in all tested samples from each fermentation medium (Fig. 1, Table 3). Particularly, high levels of lydicamycins and desferrioxamines were detected in the ΔpumI and ΔpumN mutants (Table 3; Supplementary Fig. 1).

Table 3 Relative amounts of the identified metabolites in the different pum mutants. Amounts are expressed as ratios to those observed in the WT strain in the same medium. Note that for NK1 through NK3, which are not detected in the WT, their presence is indicated with an “X”. The highest relative amounts of metabolites are in bold type.

We also detected a molecular family with a node at m/z 303 [M + 2H]2+ (Fig. 1), the HR-MS fragmentation pattern and UV spectrum of which (Supplementary Fig. 2) match those of the ureylene-containing oligopeptide antipain, a family identified used an authentic standard in previous work19. This family of compounds is produced by non-ribosomal peptide synthetases in numerous bacteria and functions as a protease inhibitor20. Consistently, we located a corresponding BGC in the ID38640 genome, that exhibits 61–78% gene sequence identity with antipain BGCs in the MIBIG database21 (Table 2). The antipain molecular family is found in both the WT strain and in most pum mutants, in both media, with highest amounts detected in the WT strain (Table 3; Supplementary Fig. 1).

A self-loop feature (Fig. 1) was detected with an exact mass corresponding to ulleungdin, a recently reported lassopeptide from Streptomyces sp. KCB13F00322 (found m/z 796.8835 [M + 2H]2+; calculated m/z 796.8859 [M + 2H]2+). The identification was consistent with the MS fragmentation pattern (Supplementary Fig. 3) and with a lassopeptide BGC (Table 2; Supplementary Fig. 3) encoding a predicted core peptide identical to ulleungdin. Ulleungdin was detected in all tested strains, in both fermentation media, with the WT strain showing trace levels (Table 3; Supplementary Fig. 1).

The molecular networking analysis of Fig. 1 was carried out using a cosine score above 0.7. This value was also used to form MS clusters. This filter excluded ectoine, a methyl, tetrahydropyrimidinecarboxylic acid that protects many bacterial species from osmotic stress, since this metabolite shows very poor fragmentation. Nonetheless, we were able to identify a peak, eluting at 1.1 min, consistent with ectoine hydrophilicity, having a matching exact mass (found m/z 143.0813 [M + H]+, calculated m/z 143.0815 [M + H]+; Supplementary Fig. 4). Consistently, we located a BGC in the ID38640 genome corresponding to the ectoine BGC (Table 2). Ectoine is detected in all tested strains (Table 3; Supplementary Fig. 1).

Additionally, the molecular network of Fig. 1 highlighted three other molecular families corresponding to unknown molecules. Four different pum knockout mutant strains, when grown in M8 medium, produced four related metabolites with m/z 536 [M + H]+, m/z 554 [M + H]+, m/z 555 [M + H]+ and m/z 642 [M + H]+ (“NK1” in Table 3; Supplementary Fig. 5). Two additional signals, m/z 558 [M + H]+ and m/z 530 [M + H]+, were observed in samples from the ΔpumG and ΔpumL strains, when grown in PumP1 medium (“NK2” in Fig. 1; Table 3; Supplementary Fig. 5). Moreover, two signals corresponding to doubly charged masses m/z 416 [M + 2H]2+ and m/z 423 [M + 2H]2+ were observed in samples from ΔpumL in M8 medium (“NK3”in Fig. 1; Table 3; Supplementary Fig. 5). No matches of these signals were found in the Dictionary of Natural Products or in the Natural Product Atlas23,24, and our internal set of 5200 Streptomyces molecular fingerprints indicated that NK1, NK2 and NK3 are rare occurrences. Thus, these molecules may represent novel metabolites worthy of further investigations.

We also observed a molecular family with m/z 259 [M + H]+ and m/z 421 [M + H]+, with exact masses of m/z 259.1089 [M + H]+ and 421.1615 [M + H]+, respectively (Supplementary Fig. 6). The associated LC peaks show a UV–Vis spectrum with absorption maxima at 254, 304 and 370 nm. These properties match those of pyridindolol and pyridindolol glucoside, produced by Streptomyces alboverticillatus and Streptomyces parvulus, respectively25,26. The MS2 fragmentations are consistent with this annotation (Supplementary Fig. 6). These metabolites are detected in almost all strains, with the ΔpumN mutant showing highest levels (Table 3; Supplementary Fig. 1).

Pyridindolol biosynthesis has not been studied previously and no BGC possibly linked to this metabolite could be found in the antiSMASH output. However, it has been reported that the β-carboline moiety present in pyridindolol is formed by a “Pictet-Spenglerase” (PSase), an enzyme that joins an amino group with an aldehyde27. The enzyme StnK2 has been shown to function as a PSase in streptonigrin biosynthesis in streptomycetes28. Accordingly, we searched the ID38640 genome for StnK2 homologs and identified QIK04791.1 as having 49% sequence identity with StnK2 (Fig. 2; Table 4). The QIK04791.1-encoding region also specifies for a FAD-binding oxidoreductase, a long-chain fatty acid-CoA ligase, an aldehyde dehydrogenase, a histidine phosphatase and a F420-dependent oxidoreductase (Table 4). While most of these sequences have no paralogs in the streptonigrin BGC, we found that a syntenic region with over 90% protein-to-protein identity (Fig. 2; Table 4) is present in the genome of the pyridindolol producer Streptomyces alboverticillatus (MUFU00000000; Fig. 2; Table 4). Based on our observations, we hypothesize that pyridindolol formation entails condensation of tryptophan with a C-3 unit, possibly glyceraldehyde(phosphate) by the PSase; aromatization of the newly formed ring by the FAD-binding- and/or the F420-dependent oxidoreductase; and reduction of the carboxyl group by the aldehyde dehydrogenase (Fig. 2). The order in which the hypothesized reactions occur awaits further analysis, as does the possible role of the conserved long-chain fatty acid-CoA ligase and histidine phosphatase present in the conserved segment. This BGC, which lies at one end of the genome sequence in Streptomyces sp. ID38640, has been added to Table2 and designated “Other0”.

Figure 2
figure2

Putative pyridindolol BGC and hypothetical roles in metabolite formation. (a) Identified region containing the PSase homolog (dark grey) from the ID38640 genome (top) and syntenic region found in the pyridindolol producer S. alboverticillatus (bottom). The percent identities between orthologs are shown, while deduced functions are reported in Table 4. (b) Pyridindolol structure, highlighting the likely building blocks tryptophan (black) and glyceraldehyde (red), and the modifications necessary, aromatization (blue) and reduction (green), for the final structure.

Table 4 The proposed pyridindolol BGC.

Overall, our work led to the identification of seven metabolite-BGC pairs (Table 2). This leaves 21 BGCs orphan of their product, including those for geosmin and methylisoborneol, volatile metabolites unlikely to be detected under our conditions, and hopene, unlikely to be present in our samples because of its lipophilicity. Thus, it remains to be determined whether these three metabolites are actually produced by Streptomyces sp. ID38640. Overall, 18 BGCs await matching metabolites and 3 identified metabolites are missing a matching BGC.

Notably, this work has demonstrated that changes in a small region of the genome facilitate the detection of additional metabolites. It has been previously reported that blocking biosynthesis of a specialized metabolite can facilitate detection of novel chemistry29. However, we are not aware of studies showing that different blocks in a single pathway can significantly alter the metabolite levels of biosynthetically unrelated metabolites. Accumulation of a particular PUM intermediate does not appear the reason for altering the metabolic profiles: for example, ectoine levels are tenfold enhanced in the ΔpumE, ΔpumJ and ΔpumK mutants that accumulate different PUM intermediates (Table 1). At the same time, not all mutants accumulating the same PUM intermediate show similar increases in ectoine levels. Thus, the observed modulation of specialized metabolite levels could result from the introduction of the apramycin resistance cassette with its strong promoter, from increased availability of precursors and/or from altered transcription by PUM. The mechanism(s) leading to altered metabolic profiles are currently unknown and further work will be necessary to establish whether this phenomenon is an oddity of the PUM pathway. Nonetheless, the generation of distinct mutants from a single BGC might be useful not only for elucidating the corresponding biosynthetic pathway (see below), but also for detecting chemistry hidden in wild type strain.

Additional insights into PUM biosynthesis

The functions of most of the pum genes and the general pathway of PUM biosynthesis have been defined from our previous in vivo experiments and bioinformatic analyses17, as summarized in Table 1. In this work, we generated knockout mutants in three additional pum genes: pumF, pumH and pumL (Table 1). PumF shows 42–45% identity to SsaA and its orthologues NpsM and PacA, which are regulators of BGC for the structurally related uridyl-peptide antibiotics sansanmycins, napsamycin and pacidamycin, respectively30. PumH, annotated as an adenylate kinase, shares 42% identity with PolQ2 and MalE from the polyoxin and malayamycin biosynthetic pathways, respectively31. PumL shares 65% identity with a NocH-like protein belonging to the major facilitator superfamily.

Replacement of pumF with the apramycin resistance gene abolished PUM production and led to the accumulation of pseudouridine (PU), amino pseudouridine (APU) and guanidine acetate (GAA) (Table 1; Supplementary Fig. 7). These results indicate that PumF is a positive regulator of PUM production that controls the conversion of APU into Gln-APU. The ΔpumL knockout mutant resulted in very low yields of PUM and accumulation of several PUM intermediates, consistent with a role of PumL in exporting the final pathway product (Table 1; Supplementary Fig. 7).

The phenotype of the ΔpumH mutant was more complex: it accumulated no PUM-related metabolite except for GAA (Table 1; Supplementary Fig. 7); and, unlike the ΔpumJ strain17, PUM production could not be rescued by adding PU to the production medium. Thus, the ΔpumH phenotype was identical to that of the previously reported ΔpumI mutant17, which likewise accumulated no intermediate except for GAA and could not convert PU into PUM. The simplest interpretation of these results is that inactivation of pumH or pumI through insertion of an apramycin resistance cassette has a polar effect on expression of downstream genes in the same transcriptional unit. Consistent with this interpretation, pumH and pumI overlap by 20 bp, and pumI and pumJ by 4-bp, suggesting they belong to a single transcriptional unit. Insertion of the apramycin resistance cassette into the kinase-encoding pumH would thus disable also the oxidoreductase PumI and the pseudouridine synthase PumJ, while insertion into pumI would leave only pumH intact. Recently, Draelos et al. have demonstrated 2′-phosphorylation of early intermediates in the nikkomycin and polyoxin pathways by enzymes belonging to the same family as PumH32, corroborating the hypothesis that intermediate phosphorylation also occurs during PUM biosynthesis.

We also assessed PUM and PUM precursors accumulated in the WT strain and in the ten pum knockout mutants in the molecular network. PUM (m/z 487 [M + H]+) appears as a single loop detected only in the WT strain and, to a lesser extent, in the ΔpumJ and ΔpumL mutants, irrespective of the cultivation medium (Fig. 1 and Table 1). We also detected a molecular family including PU, m/z 244 [M + H]+, and APU, m/z 245 [M + H]+. PU accumulates only in the ΔpumF, ΔpumG and ΔpumK mutants, while APU accumulates mainly in the ΔpumF and ΔpumK mutants, with highest levels of both metabolites observed in ΔpumK (Table 1). The late PUM intermediates Gln-APU (m/z 372 [M + H]+) and deoxy-PUM (m/z 471 [M + H]+) cluster together (Fig. 1). As expected, Gln-APU is found in samples from the ΔpumE, ΔpumL, ΔpumN and ΔpumM strains, with the latter strain accumulating the highest level. Deoxy-PUM was detected in samples from the WT, ΔpumE and ΔpumL strains, with highest level present in WT, in agreement with previous results (Fig. 1, Table 1). In a different portion of the same network we observed a signal at m/z 388 [M + H]+) consistent with N-hydroxy-Gln-APU (OH-Gln-APU), which corresponded to a hydrophilic peak with the pseudouridine-characteristic UV maximum at 263 nm. This species, which had not been noted in previous work because of its low abundance (Table 1), was detected in samples from the WT and, in lower amounts, from the ΔpumL, ΔpumM and ΔpumN strains. The levels of the PU-containing intermediates sharing the same chromophore were quantified as reported in Table 1.

The results presented here enable us to confirm and extend the previously proposed biosynthetic pathway for PUM17 (Fig. 3). During the early biosynthetic steps, the substrate for the kinase PumH may be either uridine, PU or PU aldehyde (Fig. 3), with PumJ, PumI and PumG acting sequentially for C-isomerization, alcohol oxidation and amine formation, respectively. By analogy with the nikkomycin/polyoxin pathways32, phosphorylation is likely to occur at the 2′ position, as shown in Fig. 3. Subsequently, the phosphate group is removed by PumD or by a housekeeping phosphatase. A key step in the pathway appears to be the conversion of APU into Gln-APU by PumK, a conversion controlled by the regulator PumF. The detection of OH-Gln-APU suggests that N-hydroxylation by PumE precedes addition of GAA by PumM, consistent with the well-known facilitated hydroxylation of amines with respect to amides33. In the absence of PumE, PumM uses Gln-APU as substrate, leading to deoxyPUM as a shunt metabolite. While in the absence of PumM, Gln-APU preferentially accumulates, suggesting that either conversion of Gln-APU into its hydroxyderivative is inefficient or that expression of pumE is altered in this context. Finally, PumL appears to be a transporter for PUM.

Figure 3
figure3

Revised biosynthetic pathway for pseudouridimycin. (a) PUM BGC, with established role for PumF. (b) Proposed pathway. Enzymes and intermediates reported within brackets have not been experimentally determined. X = H or PO3H2. Phosphorylation can be introduced by PumH after PU formation and before Gln addition. See text for abbreviations.

PUM production by other streptomycetes

In previous work, we identified pumJ-related sequences linked to putative BGCs in numerous microbial genomes and we predicted these BGCs specify biosynthesis of PUM or closely related metabolites17. Production of PUM has been previously demonstrated only for Streptomyces spp. ID38640 and ID38673, from the NAICONS collection14, and for Streptomyces albus DSM 4076334,35. To investigate whether the strains harboring a PUM BGC did produce PUM and other metabolites shared with Streptomyces sp. ID38640, we investigated four Streptomyces strains: S. rimosus ATCC 10970, producer of oxytetracycline; S. mobaraensis DSM 40847, producer of the NADH reductase inhibitor piericidin; S. eurocidicus ATCC 27428, producer of the antifungal polyene eurocidin; and S. flocculus DSM 40313, producer of the aminoquinone antibiotic streptonigrin. [S. flocculus has been recently reclassified36 and will be referred as S. albus DSM 40313 hereafter.] These compounds have been known for several decades and the producer strains have been investigated by several laboratories, but PUM production has to our knowledge not been observed.

When grown in a single medium and analyzed at three different time points, each Streptomyces species, in addition to the expected metabolites oxytetracycline, piericidin, eurocidin D or streptonigrin, produced PUM (Fig. 4a), at a level comparable to Streptomyces sp. ID38640 (around 200 μM) for S. rimosus and S. mobaraensis or at approximately half these levels for S. albus and S. eurocidicus. These results indicated that the PUM BGC in these species is actively expressed and that PUM can be easily detected when properly looked for.

Figure 4
figure4

Analysis of PUM production in other Streptomyces strains. (a) Extracted ion chromatograms of PUM (m/z 487 [M + H]+) and strain specific metabolites from S. rimosus, S. mobaraensis, S. eurocidicus and S. albus. (b) Complete molecular network of two samples from each of the above PUM producers and from ID38640. Strain-specific features are color coded as: ID38640, green; S. eurocidicus, pink; S. albus, orange; S. mobaraensis, red; and S. rimosus, blue. Features detected in more than one strain are in light blue. All strains were cultivated in M8 medium and samples prepared at 72 h.

We were interested in establishing the phylogenetic relationship and the extent of shared metabolites of the five PUM producers. We thus applied autoMLST37 to construct a high-resolution species tree, which revealed ten major clades and three branches formed by a single strain each (Supplementary Fig. 8). Streptomyces sp. ID38640 belongs to clade 2 while, among the PUM producers reported above, only S. rimosus (clade 7) and S. mobaraensis (single-strain branch) were picked up. A phylogenetic tree of the five PUM producers showed that Streptomyces sp. ID38640 clustered with S. rimosus, while S. eurocidicus and S. mobaraensis formed a separate clade (Supplementary Fig. 9).

The ID38640 genome does not harbor BGCs for oxytetracycline, euricidin, piericidin or streptonigrin. In addition to PUM, only three additional BGCs are shared by the five strains: those for the frequently encountered Streptomyces metabolites geosmin and hopene; and that for the BGC labeled Other5 (Table 2). The latter BGC, consisting of a syntenic region of 8 conserved ORFs, is of unknown function and has been identified by antiSMASH as siderophore.

We next investigated whether the other PUM-producing strains shared other metabolites with Streptomyces sp. ID38640, using procedures similar to those described above. The resulting molecular network, represented in Fig. 4b, contains 630 features, including media components, of which 385 (61%) are organized in 62 molecular families. As highlighted by a red rectangle, a two-member family containing PUM and deoxy-PUM is found in samples from all strains. Of the families annotated in Fig. 1, lydicamycins, pyridindolol, ulleungdin and NK1 through NK3 remain ID38640-specific. Desferrioxamines were detected in S. rimosus and S. albus, while antipain was detected in S. mobaraensis. As described above, manual inspection of the LC–MS profiles showed ectoine in extracts from S. rimosus and S. albus. Additional metabolites were dereplicated in the samples and the corresponding BGCs were identified in the strain genomes (M.I., unpublished observations) but none of these additional molecules matched unannotated metabolites detected in Streptomyces sp. ID38640.

Taken together, the above results indicate that the PUM BGC is not restricted to a specific Streptomyces clade and that PUM is not regularly co-produced with other metabolites. It will be interesting to establish whether knockout in the PUM pathway in the other PUM producers can also alter their metabolic profiles.

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